NS3 contains four regions of sequence homology to trypsin-like serine proteases. Two of these four regions in NS3 ("boxes" 3 and 4) contain residues predicted to be important for substrate binding (aa 1604, 1605, 1625, 1627 and 1628). We mutagenized some of these residues in DEN2 to characterize their requirement for protease activity and to identify mutants with partial cleavage defects. In all, 46 mutations were analyzed. Mutations in box 4 generally abolish protease activity, consistent with the hypothesis that box 4 forms part of the substrate-binding pocket. In contrast, many of the mutations in box 3, including those in aa 1604 and 1605, retain significant protease activity. Other mutations in box 3, clustered around the catalytic serine, abolish cleavage. This confirms that some resisues in box 3 are important for protease function, but is inconsistent with the proposed role for aa 1604 and 1605 in substrate binding. Work is underway to introduce some of the mutations with partial activity back into DEN2, in an effort to confirm the observed effect on protease function and possibly to attenuate the virus. A major portion of the regulatory responsibilities of LVVD includes the evaluation of INDs for the development of live virus vaccines to protect against diseases caused by flaviviruses (e.g., dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), alphaviruses (e.g., Chikungunya virus, Eastern and Venezuelan equine encephalitis viruses), and arenaviruses (e.g., Junin virus, Hantaviruses). These are all positive-stranded RNA viruses. Thus, a viable strategy for producing a vaccine against any of these viruses is to identify potential attenuating mutations and engineer them into the viral genome using an infectious cDNA clone. Moreover, the principles applied to the identification and testing of putative vaccine strains are identical, regardless of the technology employed to derive them. Thus, the work described in this project will be invaluable in helping us to critique methods used by others to derive vaccine viruses and to evaluate them. In addition, we seek to develop our own attenuated dengue viruses as candidates for vaccine development.